1. Field of the Invention
The present invention relates to an electrochemical test device suitable for determining the presence or concentration of chemical and biochemical components (analytes) in aqueous fluid samples and body fluids such as whole blood or interstitial fluid. Additionally, this invention relates to a method of using such test devices for determining the presence or concentration of an analyte and to processes for preparing such a test devices.
2. State of the Art
Medical studies have demonstrated that the incidence of serious complications resulting from diabetes, such as vision loss and kidney malfunction, can be significantly reduced by careful control of blood glucose levels. As a result, millions of diabetics use glucose testing devices daily to monitor their blood glucose concentrations. Additionally, a wide variety of other blood testing devices are used to determine the presence or concentration of other analytes, such as alcohol or cholesterol, in aqueous samples, such as blood.
Such blood testing devices typically employ either a dry chemistry reagent system or an electrochemical method to test for the analyte in the fluid sample. In recent years, electrochemical testing systems have become increasingly popular due to their small size and ease of use. Such electrochemical testing systems typically use electrochemistry to create an electrical signal which correlates to the concentration of the analyte in the aqueous sample.
Numerous electrochemical testing systems and related methods are known in the art. For example, European Patent Publication No. 0 255 291 B1, to Birch et al., describes methods and an apparatus for making electrochemical measurements, in particular but not exclusively for the purpose of carrying out microchemical testing on small liquid samples of biological, e.g. of clinical, origin.
European Patent Publication No. 0 351 891 B1, to Hill et al., teaches a method of making an electrochemical sensor by printing. The sensor is used to detect, measure or monitor a given dissolved substrate in a mixture of dissolved substrates, most specifically glucose in body fluid.
U.S. Pat. No. 5,391,250, to Cheney II et al., teaches a method of fabricating thin film electrochemical sensors for use in measuring subcutaneous or transdermal glucose. Fabrication of the sensors comprises placing a thin film base layer of insulating material onto a rigid substrate. Conductor elements for the sensors are formed on the base layer using contact mask photolithography and a thin film cover layer.
U.S. Pat. No. 5,437,999, to Diebold et al., teaches a method of fabricating thin film electrochemical devices which are suitable for biological applications using photolithography to define the electrode areas. The disclosures of each of the above patent specifications are incorporated herein by reference in their entirety.
An excellent reference on materials and process for fabricating electronic components is Charles A. Harper, Handbook of Materials and Processes for Electronics, 1984, Library of Congress card number 76-95803. It provides detail process information on thick film, thin film and photo resist processes.
Existing electrochemical testing systems, however, have certain limitations from the perspective of the end user or the manufacturer. For example, some electrochemical testing systems are difficult or costly to manufacture. As a result, such devices are too expensive to be used on a daily basis by, for example, diabetics. Other electrochemical testing systems are not sufficiently accurate to detect certain analytes at very low concentrations or to give reliable measurements of the analyte""s concentration. Additionally, many electrochemical devices are too large to be easily carried by those needing to test their blood on a regular basis throughout the day. Thus, a need exists for improved electrochemical test devices.
The present invention utilizes amorphous semiconductor applied with thin film manufacturing techniques and membranes which have a skin on each planar surface to provide an electrochemical test device suitable for determining the presence or concentration of analytes in aqueous fluid samples. By using amorphous semiconductor materials applied with film manufacturing techniques and dual skin membranes, uniform electrochemical test devices having well-defined reproducible electrode areas can be manufactured economically.
In particular, the test devices of this invention have very uniform surface areas which reduce the variability of the electrochemical test. In this regard, it has been found that the surface area of the electrodes and the accuracy of applying the reagent are critical to producing an accurate test. If the surface area is not consistent from test to test then each of the test devices must be individually calibrated to insure accurate readings. The test devices of the present invention permit highly accurate electrochemical analyte measurements to be performed on very small aqueous fluid samples without the need for individual calibration of each test device. The present inventions provide for the accurate reproduction of the test devices by using controlled deposition methods, such as sputtering or vapor deposition and smooth skin membranes to form the electrodes with consistent size and surface morphology from device to device in continuous production. These devices can also be readily manufactured due to the lower cost and the flexible nature of the amorphous semiconductor materials which facilitates production by continuous roll processing versus the step and repeat printing methods currently employed. The ability to use continuous processing to fabricate the device, such as continuous processes utilizing continuous roll coating, continuous roll sputtering, continuous systems utilizing contact masks, results in high volume manufacturing capability and substantial cost reductions over the step and repeat processes. Additionally, the amorphous nature of the conductors electrodes and constructed and used according to this invention eliminates problems found in prior test devices which utilize conventional conductor and semiconductor materials, which are crystalline in nature or are noble metals and, as a result, require flat and rigid substrates to prevent cracking during manufacture, distribution or use. The membrane in the present invention is the earlier for the indicating reagent and forms the surface for the electrode formation. The membrane comprises the matrix in which the reagent is carried or impregnated and comprises the two exterior skin surfaces on which the electrodes are placed. The skin surfaces can be smooth skin suitable for carrying the electrodes and can be porous to pass the aqueous fluid samples or can have pores sized to screen or block selected components from aqueous fluid sample, such as red blood cells in a blood sample.
Dry electrochemical test devices fall into two primary configurations. The first configuration utilizes two electrodes, i.e., a working electrode and a counter electrode. The second configuration utilizes three electrodes, i.e., a working electrode, a counter electrode and a reference electrode. The use of the reference electrode and a reference material provides a fixed reference for the test. The test devices of the present invention are well suited for a two electrode system however, a contact mask could be employed during sputtering to create a surface with two electrodes.
Accordingly, in one of its aspects, the present invention provides an electrochemical test device for determining the presence or concentration of an analyte in an aqueous fluid sample, said electrochemical test device comprising:
(a) a nonconductive surface;
(b) a working electrode comprising an amorphous semiconductor material affixed to the non-conductive surface of a double skin membrane, said working electrode having an first electrode area, a first lead and a first contact pad;
(c) a counter electrode comprising an amorphous semiconductor material affixed to the opposite nonconductive surface of a double skin membrane, said counter electrode having an second electrode area, a second lead and a second contact pad; and
(d) a reagent capable of reacting with the analyte to produce a measurable change in potential which can be correlated to the concentration of the analyte in the fluid sample, said reagent being imbibed into the membrane matrix between the two electrode surfaces.
In another embodiment of this invention, the test device further comprises a reference electrode comprising an amorphous semiconductor material affixed to the non-conductive surface, said reference electrode having an electrode area, a lead, and a contact pad, and wherein at least a portion of the electrode area is overlaid with a reference material. Preferably, the reference material is a silver/silver chloride layer, a mercury/mercury chloride layer or a platinum/hydrogen material. This electrode could be either the counter electrode or an independent third electrode.
In a preferred embodiment of this invention, the test device further comprises a blood separating membrane with two skin surfaces. The blood separating membrane separates whole blood samples into highly colored and relatively clear fluid portions before analysis. The blood separating membrane effectively blocks or filters red blood cells and allows essentially clear fluid to pass to the test reagent imbibed in the membrane matrix. As a result, the analyte is measured in the clear fluid portion of the sample contacting the electrodes thereby substantially eliminating the red blood cells from the reaction and minimizing interference from the cells present in blood. This embodiment has the additional benefit of keeping the test site from drying out and thereby improves the performance of test devices designed for small sample sizes, such as in the 1 to 5 microliter range.
Preferably, the membrane is selected from polysulphone, polyethersulphone, or nylon and is cased with a tight pore skin on each side and a relatively isotropic matrix between each skin surface
The skin pore size is relatively tight approximately 0.1 to 0.5 microns in size and the isotropic matrix being 0.5 microns or greater in pore size. The tight pore size provides a uniform surface morphology on which the amorphous semiconductor electrodes are formed according to this invention. Better surface morphology of the membrane results in a more consistent surface for the amorphous semiconductor electrodes. This provides improved accuracy of test results and consistency of performance.
Preferably, the amorphous semiconductor material used in this invention is amorphous silicon oxide. More preferably, the amorphous silicon oxide is doped with lithium, potassium, or a similar conducting ion to increased conductivity. Doping with lithium is particularly preferred. However, amorphous carbon, gold, silver or other conductor materials which do not interfere with the electrochemistry of the reagent system are also suitable. The amorphous semiconductor material can be applied by using various techniques including sputtering, evaporation, vapor phase deposition or other thin film deposition technique to form a conductive layer on the membrane surface The surface texture of the amorphous semiconductor material is preferably less than 13 micro inches or 0.33 microns. However, rougher textures can be used depending on the accuracy of the desired test device.
The reagent employed in the electrochemical test device is typically selected based on the analyte to be tested and the desired detection limits. The reagent preferably comprises an enzyme and a redox mediator. When the analyte to be detected or measured is glucose, the enzyme is preferably glucose oxidase and the redox mediator is potassium ferrocyanide.
The electrochemical test device of the present invention is used to determine the presence or concentration of an analyte in an aqueous fluid sample. Accordingly, in one of its method aspects, the present invention provides a method for determining the presence or concentration of an analyte in an aqueous fluid sample, said method comprising:
(a) providing an electrochemical test device comprising: (i) double skinned membrane; (ii) a working electrode comprising an amorphous semiconductor material affixed to the membrane surface, said working electrode having an first electrode area, a first lead and a first contact pad area; (iii) a counter electrode comprising an amorphous semiconductor material affixed to the non-conductive surface, said counter electrode having a second electrode area, a second lead, and a second contact pad; and (iv) a reagent capable of reacting with the analyte to produce a measurable change in potential which can be correlated to the concentration of the analyte in the fluid sample, said reagent being imbibed into the membrane matrix between the two electrode surfaces;
(b) inserting the electrochemical test device into a meter device;
(c) applying a sample of an aqueous fluid to the membrane area of the working electrode;
(d) reading the meter device to determine the presence or concentration of the analyte in the fluid sample.
In another embodiment, the test device employed in this method further comprises a reference electrode comprising an amorphous semiconductor material affixed to the counter electrode membrane surface, said reference electrode having a third electrode area, a third lead, and a third contact pad, and wherein at least a portion of the third electrode area is overlaid with a reference material.
Preferably, the reference material is a silver/silver chloride layer, a mercury/mercury chloride layer or a platinum/hydrogen material. Silver/silver chloride is a particularly preferred reference material. The separation of the counter electrode conductive path and reference electrode is accomplished by using a mask to create the different geometries on the same surface.
Preferably, the membrane is selected from polysulphone, polyethersulphone, or nylon and is cased with a tight pore skin on each side and a relatively isotropic matrix between each skin surface
The skin pore size is relatively tight approximately 0.1 to 0.5 microns in size and the isotropic matrix being 0.5 microns or greater in pore size. The tight pore size provides a uniform surface morphology on which the amorphous semiconductor electrodes are formed according to this invention. Better surface morphology of the membrane results in a more consistent surface for the amorphous semiconductor electrodes. This provides improved accuracy of test results and consistency of performance.
As discussed above, the present invention utilizes amorphous semiconductor materials and thin manufacturing techniques to provide electrochemical test devices. Thin film technologies can be used to create the amorphous semiconductor material conductive layers and electrodes according to this invention. Accordingly, in one of its process aspects, the present invention provides a process for preparing an electrochemical test device suitable for determining the presence or concentration of an analyte in an aqueous fluid sample, said process comprising the steps of:
(a) providing a skinned membrane having a first and an opposite second surface;
(b) depositing an amorphous semiconductor material on said first surface to form a conductive layer,
(c) depositing an amorphous semiconductor material on said opposite second surface to form a conductive layer
(d) applying a reagent to the membrane which is imbibed into the membrane matrix between the two surfaces, said reagent being capable of reacting with an analyte in an aqueous fluid sample to produce a measurable change in potential which can be correlated to the concentration of the analyte in the fluid sample.
In another embodiment, step (c) of this process further comprises forming a reference electrode comprising a third electrode having a third electrode area, a third lead and a third contact pad. This is accomplished by using a mask to form the two distinct electrodes and in a step (e) forming a silver chloride surface.
In a preferred embodiment, step (a) above comprises the steps of:
(f) providing a double skinned membrane with the correct pore size distribution; and
In another preferred embodiment, step (d) above comprises the steps of:
(h) positioning a mask on the opposing membrane surface and sputtering an amorphous conductive surface to the membrane surface to form independent electrodes;
(i) said second exposed conductive surface area comprising (i) a counter electrode comprising a second electrode having a second electrode area, a second lead and a second contact pad, and optionally (ii) a reference electrode comprising a third electrode having a third electrode area, a third lead and a third contact pad.
In further preferred embodiment, step (d) above further comprises the steps of:
(o) positioning a second mask on the opposing surface so that the opposing electrode area is masked and the third reference electrode area is exposed;
(p) applying a reference material the third electrode area;
(q) removing the mask.
Preferably, the process employed to prepare the test devices of this invention is a continuous process. The ability to use continuous processing to fabricate the test devices, such as a continuous process utilizing continuous roll coating, continuous roll sputtering, continuous sputtering systems utilizing contact masks, results in high volume manufacturing capability and substantial cost reductions over the step and repeat printing processes.